Mycobacterium tuberculosis vaccine

ABSTRACT

The present invention relates to the use of live mycobacterium of the  M. tuberculosis  complex for preparing a medicament, wherein the function of the zmp1-gene is inactivated, pharmaceutical compositions prepared from such mycobacteria as well as a method for the treatment and/or prophylaxis of a disease or medical condition using said pharmaceutical composition.

FIELD OF THE INVENTION

The present invention relates to the use of live mycobacterium of the M.tuberculosis complex for preparing a medicament, wherein the function ofthe zmp1-gene is inactivated, pharmaceutical compositions prepared fromsuch mycobacteria as well as a method for the treatment and/orprophylaxis of a disease or medical condition using said pharmaceuticalcomposition.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis is one of the most successful pathogens knowntoday, killing millions of individuals worldwide every year. A hallmarkof M. tuberculosis infection is that following phagocytosis themicroorganisms resist lysosomal delivery, instead residing withinphagosomes that do not fuse with lysosomes. Phagolysosomes are equippedwith the machinery to generate peptide—MHC II complexes. Inhibition ofphagolysosome fusion has been proposed to represent a mechanism by whichM. tuberculosis escapes efficient antigen presentation by host MHC IIcomplexes. In addition, cross-presentation of peptides derived fromparticulate antigens and exploiting the conventional MHC I pathway canoccur via a putative phagosome-to-cytosol mechanism. Alternatively, thiscan occur by fusion and fission of phagosomes with endoplasmicreticulum-derived vesicles containing newly synthesized MHC I molecules.

BCG is a live attenuated vaccine derived from M. bovis back in 1919.More than three billion doses of the vaccine have been administeredworldwide. Although BCG is relatively safe and inexpensive, its efficacyis highly variable (Fine, P. E. M., Lancet 346, 1339-1345, 1995). Thereasons for the varying efficacy of BCG in protection againsttuberculosis are poorly understood. One explanation builds on theobservation that BCG has lost important genes during the laboratoryattenuation process (Behr et al., Nature 389, 133-134, 1997). A greatdeal of work has been done to improve—with varying success—the efficacyof BCG by introducing additional copies of existing genes (Horwitz etal., PNAS, USA 97, 13853-13858, 2000) or by reintroducing some of thegenes that were lost during the in vitro attenuation process (Pym etal., Nat. Med. 9, 533-539, 2003).

Alternative live vaccination strategies focus on attenuated M.tuberculosis. It is commonly assumed that tuberculosis disease willprotect, at least partially, against subsequent reinfection. However,the failure of natural disease to protect against re-infection diseaseat a later point indicates that immunity evoked by natural infection islimited, partially explaining the relative ineffectiveness ofvaccination with BCG. The limited post-infectious immunity may alsoindicate that M. tuberculosis actively escapes immune surveillance.

Recently, the present inventors reported that a putative mycobacterialzinc metallo-protease, Zmp1, may play an important role in diseasepathogenesis by interfering with two pathways of pathogen defense:inflammasome activation and phagosome maturation (Master et al.,Mycobacterium tuberculosis prevents inflammasome activation. Cell HostMicrobe 3, 224-232, 2008).

It is the object of the present invention to provide a livemycobacterium-based medicament with increased immunogenicity andimproved protective efficacy.

This object is solved by the use of at least one live mycobacterium ofthe M. tuberculosis complex for preparing a medicament, wherein thefunction of the zmp1-gene is at least partially inactivated, preferablyinactivated.

It was found that live mycobacteria, wherein the function of thezmp1-gene is at least partially inactivated, preferably substantially orfully inactivated, elicit an increased immunogenicity and protectiveefficacy when compared to zmp1 active mycobacteria.

The term live mycobacterium of the M. tuberculosis complex as usedherein refers to a mycobacterium species or strain which is a member ofthe M. tuberculosis complex, which includes but is not limited to M.tuberculosis, M. bovis BCG, M. bovis, M. africanum and M. microti.

Preferably, a mycobacterium of M. bovis, preferably M. bovis BCG, or M.tuberculosis is used for preparing a medicament according to theinvention.

The mycobacterium used for the invention is alive, i.e. capable ofpropagation in a host, in particular in a mammalian host, preferably ina human host.

It is self-evident, that live mycobacteria for medical use must beattenuated to a degree not harmful to patients in need thereof. Hence,the mycobacterium used for the invention is preferably non-virulent,i.e. the genes responsible for virulence have been inactivated, and doesnot evoke or at least evokes minor disease symptoms of a mycobacterialinfection in a mammal, preferably human.

Mycobacteria of the M. tuberculosis complex produce endogenous antigenswhich are cross-reactive with M. tuberculosis. Antibodies raised againstsuch cross-reactive antigen will also bind specifically to one or moreantigens from M. tuberculosis and are capable of evoking and/orpotentiating an immune response against M. tuberculosis infection in amammal. For example, cross reactive antigens of M. bovis BCG will evokeand/or potentiate an immune response against M. tuberculosis.

Mycobacteria of the M. tuberculosis complex are not only useful forexpressing cross-reactive antigens but also have applications as adelivery system for the expression of exogenous or foreign antigensand/or immunogens. The efficacy of this delivery system lies in the longpersistence in the immunized host (Stover et al., Nature, 351, 456-460,1991; Aldovini and Young, Nature, 351, 479-482, 1991).

Exemplary suitable antigens for delivery via mycobacteria of the M.tuberculosis complex include viral, protozoal, tumour cell-derived,bacterial and fungal antigens. For example, antigens derived from H.pylori, measles virus, mumps virus, rubeola virus, B. burgdorferi (e.g.OspA), herpes virus, papilloma virus, Pneumococcus spp (e.g. surfaceprotein A), tumour cells, leishmania (e.g. surface proteinase gp63), HIVor SIV may be used. Such an antigen may be useful in the treatment ofulcers, measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus,diphtheria, leishmaniasis or AIDS.

In a preferred embodiment, mycobacteria for use in the present inventionmay also comprises genetic material encoding an antigen and/or immunogenexogenous or foreign to the mycobacterium. More preferably, theexogenous or foreign antigen and/or immunogen is selected form the groupconsisting of viral, protozoal, tumor cell-derived, bacterial and fungalantigens and immunogens, preferably selected from the group consistingof antigens and/or immunogens from H. pylori, measles virus, mumpsvirus, rubeola virus, B. burgdorferi, preferably protein ospA of B.burgdorferi, herpes virus, papilloma virus, Pneumococcus spp, preferablyprotein A of Pneumococcus spp., tumour cells, leishmania, preferablysurface proteinase gp63 of leishmania, HIV and SIV.

Hence, the mycobacterium for use in preparing a medicament according tothe invention is useful for the prophylaxis and/or treatment of diseasesor medical conditions affected by antigen and/or immunogen expression ofthe mycobacterium. An antigen evokes antibody production in a host, animmunogen affects the immune system of a host.

Most evident as well as most preferred, the medicaments prepared fromzmp1-inactivated mycobacteria according to the invention are useful forthe prophylaxis and/or treatment of mycobacterial infections, preferablyan infection of M. tuberculosis.

In a further preferred embodiment the present invention is directed tothe use according to the invention for the prophylaxis and/or treatmentof a disease or medical condition selected from the group consisting ofulcers, measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus,diphtheria, leishmaniasis and AIDS.

One or more live mycobacteria species and/or strains with an at leastpartially inactivated zmp1-gene function can be used for preparing amedicament according to the invention.

The term “function of the zmp1-gene” as used herein is intended to meanthe function of the putative mycobacterial zinc metalloprotease Zmp1 ininflammasome activation, caspase-1-dependent activation and secretion ofIL-1β as well as phagosome maturation in mammalian, in particular humanmacrophages.

Hence, the function of the zmp1-gene in a mycobacterium can be directlyverified and/or quantified by investigating inflammasome activation,caspase-1-dependent activation and/or secretion of IL-1β and/orphagosome maturation in mammalian, in particular human macrophages uponinfection with the mycobacterium of interest. Of course, other directand indirect effects of protein Zmp1, e.g. effects resulting from IL-1βactivation and/or secretion, are also indicative of the function of thezmp1 gene and may also be used instead of or in addition to the abovedirect indicators.

The term “at least partially inactivated”, as used herein, is meant toindicate a loss of zmp1 gene function directly and/or indirectlyattributable to the expression of the zmp1 gene. Of course, theinactivation and extent of inactivation can best be determined in directcomparison to a mycobacterium of the same strain under identicalexperimental conditions but comprising a fully functional and expressedzmp1 gene. Preferably, “at least partially inactivated” relates to aloss of more than 10%, preferably more than 50%, more preferably morethan 70%, most preferably more than 90% of a function directly and/orindirectly attributable to the expression of the zmp1 gene in amycobacterium.

Partial and/or full inactivation of genes in mycobacteria of the M.tuberculosis complex for use in the present invention can be achieved byany conventional method in the art of gene mutation, e.g. recombinantinsertion, replacement, deletion, frameshift and/or homologousrecombination. Suitable specific methods for partially and/or fullyinactivating genes, in particular the zmp1 gene in mycobacteria aredescribed in WO 02/50262 (recA mutants), Master et al., supra, and theexamples below.

It was found that the threshold for the induction of an immune responseto M. tuberculosis antigens in immunized mammals was ten times less formammals immunized with zmp1-deficient mycobacterial strains whencompared to the threshold necessary for inducing the immune response inmammals immunized with the corresponding wild type, i.e. zmp1 activated,mycobacterial strain. Consequently, medicaments prepared frommycobacteria according to the invention evoke a substantially increasedimmunogenicity associated with increased proliferative and cytokinesecreting immune responses.

Furthermore, the protective efficacy of mammals immunized with amedicament, i.e. pharmaceutical composition/vaccine, prepared accordingto the invention against M. tuberculosis challenge was demonstrated tobe clearly superior over the protective efficacy achieved in mammalsimmunized with the corresponding wild type mycobacterial strain. Incomparison, mammals immunized according to the invention demonstratedupon M. tuberculosis challenge a more pronounced and more frequentlymphocytic infiltration into lungs.

Last but not least, increased immunogenicity and protective efficacy ofmedicaments prepared according to the invention manifests itself in asubstantially prolonged mean survival time of immunized mammals uponchallenge with virulent M. tuberculosis.

Consequently, the medicaments prepared according to the inventionpresent a significant improvement for therapeutic, prophylactic andother medical or veterinary applications of mycobacteria for deliveringendogenous, exogenous and/or foreign antigens and/or immunogens to amammal in need thereof.

A second aspect of the present invention concerns pharmaceuticalcompositions comprising as active substance at least one livemycobacterium of the M. tuberculosis complex, wherein the function ofthe zmp1-gene is at least partially inactivated, preferably inactivated,optionally and preferably combined with pharmaceutically acceptableconventional excipients and/or carriers.

to A pharmaceutical composition according to the present invention isany composition, preferably a vaccine, formulated for direct medicaladministration to a patient in need thereof, preferably a mammal, morepreferably a human.

Preferably, the mycobacterium in the pharmaceutical composition is M.bovis, preferably M. bovis BCG, or M. tuberculosis. It is also preferredthat the mycobacterium used is non-virulent.

In a preferred embodiment the pharmaceutical composition according tothe invention is one, wherein the mycobacterium comprises geneticmaterial encoding an antigen and/or immunogen exogenous or foreign tothe mycobacterium, wherein the antigen and/or immunogen is preferablyselected form the group consisting of viral, protozoal, tumorcell-derived, bacterial and fungal antigens and immunogens, morepreferably selected from the group consisting of antigens and/orimmunogens derived from H. pylori, measles virus, mumps virus, rubeolavirus, B. burgdorferi, preferably protein ospA of B. burgdorferi, herpesvirus, papilloma virus, Pneumococcus spp, preferably protein A ofPneumococcus spp., tumour cells, leishmania, preferably surfaceproteinase gp63 of leishmania, HIV and SIV.

Preferably, the pharmaceutical composition according to the invention isone suitable for the prophylaxis and/or treatment of a disease ormedical condition affected by antigen and/or immunogen expression of themycobacterium, more preferably the prophylaxis and/or treatment ofmycobacterial infections, preferably an infection of M. tuberculosis,also more preferably for the prophylaxis and/or treatment of a diseaseor medical condition selected from the group consisting of ulcers,measles, mumps, rubeola, lyme disease, herpes, cancer, tetanus,diphtheria, cancer, leishmaniasis and AIDS.

METHODS OF USE

The medicaments, i.e. pharmaceutical compositions, vaccines, etc. areuseful for a method for medical treatment and/or prophylaxis.Consequently, in a further aspect the present invention relates tomethod for the treatment and/or prophylaxis of a disease or medicalcondition comprising the step of administering a pharmaceuticalcomposition according to the invention to a mammalian, preferably ahuman patient in need thereof.

In a preferred aspect, the present invention is directed to the abovemethod, wherein the disease and/or medical condition is selected fromthe group consisting of mycobacterial infections, preferably aninfection of M. tuberculosis, ulcers, measles, mumps, rubeola, lymedisease, herpes, cancer, tetanus, diphtheria, cancer, leishmaniasis andAIDS.

For therapeutic and/or prophylactic use the pharmaceutical compositionsof the invention may be administered in any conventional dosage form inany conventional manner. Routes of administration include, but are notlimited to, intravenously, intramuscularly, subcutaneously,intranasally, intrasynovially, by infusion, sublingually, transdermally,orally, topically, or by inhalation. The preferred modes ofadministration are subcutaneous, intravenous and intranasal.

The mycobacteria may be administered alone or in combination withadjuvants that enhance stability and/or immunogenicity of themycobacteria, facilitate administration of pharmaceutical compositionscontaining them, provide increased dissolution or dispersion, increasepropagative activity, provide adjunct therapy, and the like, includingother active ingredients.

As mentioned above, pharmaceutical dosage forms of the mycobacteriadescribed herein include pharmaceutically acceptable carriers and/oradjuvants known to those of ordinary skill in the art. These carriersand adjuvants include, for example, ion exchangers, alumina, aluminiumstearate, lecithin, serum proteins, buffer substances, water, salts,electrolytes, cellulose-based substances, gelatine, water, pretrolatum,animal or vegetable oil, mineral or synthetic oil, saline, dextrose orother saccharide and glycol compounds such as ethylene glycol, propyleneglycol or polyethylene glycol, antioxidants, lactate, etc. Preferreddosage forms include tablets, capsules, solutions, suspensions,emulsions, reconstitutable powders and transdermal patches. Methods forpreparing dosage forms are well known, see, for example, H. C. Ansel andN. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems,5^(th) ed., Lea and Febiger (1990) and, in particular, Pastoret et al.,Verterinary Vaccinology, Elsevier March 1999). Dosage levels andrequirements are well-recognized in the art and may be selected by thoseof ordinary skill in the art from available methods and techniquessuitable for a particular patient. As the skilled artisan willappreciate, lower or higher doses may be required depending onparticular factors. For instance, specific doses and treatment regimenswill depend on factors such as the patient's general health profile, theseverity and course of the patient's disorder or disposition thereto,and the judgment of the treating physician.

For example, the pharmaceutical composition of the present invention canbe administered the same way as conventional pharmaceutical compositionsbased on live mycobacteria or based on other bacteria.

In a preferred embodiment, a pharmaceutical composition comprises livebacteria of M. bovis BCG zmp1.

More preferably, the mycobacteria are lyophilized and stabilized withtypical stabilizing agents such as sugars, e.g. sucrose, and/orgelatins. The pharmaceutical composition may be formulated into tabletsor other solid forms and may be later reconstituted with water into asolution for administration, e.g. orally, by injection or intranasally.

It is emphasized that the pharmaceutical compositions of the presentinvention may be administered in lower dosages than conventionalpharmaceutical compositions based on live mycobacteria because themycobacteria used by the present invention are more immunogenic andevoke an improved protective efficacy when compared to compositions withconventional mycobacteria, e.g. wild type M. bovis BCG.

In the following the present invention will be illustrated in moredetail with reference to specific embodiments which are not to beinterpreted as limiting the scope of the invention as defined by theappended claims.

FIGURES

FIG. 1: Groups of C57BL/6 mice were immunised with 100, 1000 or 10000CFU BCG WT or BCG zmp1 and challenged intradermally with PPD in thefootpad on day 21. Footpad DTH was then measured after 48 hrs.

FIG. 2: Groups of C57BU6 were immunised with 1000 BCG WT (open bars) orBCG zmp1 (filled bars). After 4 weeks, splenocytes were restimulated invitro with PPD, or Ag85A or TB10.3 peptides. IFN-g secretion (ELISA) andproliferation (3H-thymidine incorporation) were measured on day 3 and 4,respectively. * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney).

FIG. 3: Analysis of IFN-g producing CD4 and CD8 positive T cellsfollowing vaccination of mice with BCG WT, BCG zmp1 or saline (untr).Splenocytes were restimulated with PdBu or PPD and intracellular IFN-ganalysed by flow cytometry. (A) Histograms show the frequency ofIFN-g-producing CD8 T cells (mean±s.e.). (B, C) Box plots show thefrequency of IFN-g-producing CD8 (B) or CD4 (C) T cells. The boxes showthe 25^(th) and the 75^(th) percentile as wells as median, while thewhiskers show the 10^(th) and the 90^(th) percentiles; one out of tworepresentative experiments is shown.

FIG. 4: BCG zmp1 protective efficacy in a mouse mortality model. Groupsof mice vaccinated with BCG, BCG zmp1 or untreated (control) werechallenged with M. tuberculosis and survival was monitored.

EXAMPLES Example 1 Materials and Methods Mycobacterial Strains andGrowth Conditions

Mycobacterium bovis BCG were grown on Middlebrook 7H10 agar platessupplemented with oleic acid albumin dextrose catalase (OADC) (BectonDickinson), on Dubos agar, in liquid 7H9 medium supplemented with OADCin the presence of Tween 80 (0.05%) or in Dubos broth and incubated at37° C.

The construction of the BCG zmp1 knock-out mutant has been describedpreviously (Master et al., supra). Briefly, a 4.4 kbp Noll fragmentcomprising M. tuberculosis zmp1 (Rv0198c) and its flanking region wasisolated from BAC clone Rv165 (Brosch et al. Infect. Immun. 66,2221-2229 1998), parts of zmp1 (770 bp) were deleted by NheI digestionand substituted by a kanamycin resistance cassette. The resultingsuicide vector was transformed into BCG strain 1721, selection was doneon 7H10 agar supplemented with OADC in the presence of appropriateantibiotics (Sander et al., rpsL+: a dominant selectable marker for genereplacement in mycobacteria. Mol. Microbiol. 16, 991-1000, 1995).Replacement of chromosomal zmp1 locus by the inactivated allele wasdemonstrated by Southern blot analysis.

Immunisation of Mice for Analysis of DTH, Splenocyte Proliferation,Cytokine Secretion and FACS

Mice were immunised with different doses of BCG or BCG zmp1 bysubcutaneous injections. The inoculum was diluted in buffered saline andthe injection volume was 100 μl. After three weeks, the mice werechallenged by injection of 5 μg tuberculin purified protein derivativePPD (Statens Serums Institut, Copenhagen, Denmark), dissolved in 50 μlsaline, into the plantar side of the hind right footpad. Two days later,DTH reaction was analyzed by measuring the swelling of the footpad usinga spring-loaded digital micrometer (Mitutoyo, Kawasaki, Japan).

For in vitro analysis of splenocyte proliferation and cytokinesecretion, mice were euthanized and the spleens harvested four weeksafter immunisation. Briefly, 6×10⁵ RBC-free splenocytes were incubatedin round-bottom 96-well culture plates with PPD, or the M.tuberculosis-derived antigens Ag85A (LTSELPGWLQANRHVKPTGS) and TB10.3(GTHESNTMAMLARDG) in supplemented RPMI medium. After three days,supernatants were collected and frozen down for later analysis of IFN-γsecretion by ELISA (R&D Systems, Abingdon, United Kingdom). Theremaining cells were pulsed with 1 μCi ³H-labelled thymidine another 16hours for analysis of proliferation by β-scintillation.

For FACS analysis mice were primed and boosted at four week intervals.Seven days later, the splenocytes were harvested for analysis of IFN-γsynthesis by flow cytometry. Single-cell suspensions of approximately2×10⁶ RBC-free splenocytes were re-stimulated in 24-well plates and for5 hours with 5 μg/ml PPD or 0.5 μg/ml phorbol 12,13-dibutyrate (PdBu;Sigma, Buchs, Switzerland) in the presence of 5 μg/ml Brefeldin A(Sigma). The cells were then washed, incubated on ice for five minuteswith anti-CD16/CD32 in PBS/FCS 2% with 0.01% sodium azide forFc-receptor blocking, and surface stained with anti-CD4 (FITC) andanti-CD8 (PerCP) antibodies on ice for 20 minutes. After fixation inprotein-free PBS/PFA 1% for 10 minutes, and permeabilisation in PBS/NP400.1% for 3 minutes, the cells were stained for intracellular IFN-γ (APC)in PBS/FCS 2% and on ice for 35 minutes. The samples were acquired on aFACSCanto and analysed using the FACSCanto Diva software from BDBiosciences (San Jose, Calif.).

Vaccination Experiments

Female mice 8-10 weeks of age at the beginning of the experiment wereused. Clump-free mid-log phase mycobacterial suspensions were generatedand the inocula were estimated directly by microscopic examination offiltered mycobacterial suspensions in a conventional hemocytometer andadjusted to the desired concentrations. Enumeration of the inocula'sbacilli was done by plating 3-fold serial dilutions and scoringmicro-colonies and visible colonies after 3 and 20 days incubation at37° C., respectively, confirming that the accuracy of CFU countestimation by microscopy was >90%. Mice were vaccinated by subcutaneousinoculation of 10⁶ CFU of BCG, BCG zmp1 or left untreated (salinecontrol). Six weeks after vaccination mice were challenged with 10² CFUof virulent M. tuberculosis H37Rv by aerosol infection using aninhalation exposure system (Glas-Col, Terre Haute, Ind.). To assessmycobacterial loads in spleens and lungs, 0.1 ml of serial 10-folddilutions of whole organ homogenates from four individual mice per timepoint were plated onto agar, and colonies were counted after 18-20 daysof incubation at 37° C. For survival curves 6-9 mice per group wereused. Moribund animals were sacrificed.

Histopathology

Twenty two weeks after infection with M. tuberculosis, lung tissues wereexamined for pathology. Lung tissue (the middle right lobe) was frozenusing a −60° C. to −20° C. temperature gradient in an electroniccryotome and serial 6-8 μm-thick sections were obtained across thewidest area of the lobe. Sections were stained with hematoxylin andeosin and examined by an experienced pathologist.

Statistical Analyses

Non-parametric data were analysed using a two-sided Mann Whitney U testfor two independent samples or a Kruskal-Wallis H test with Dunn's posttest for three or more samples. A two-way analysis of variance (GLMunivariate analysis of variance) was performed, with footpad swelling,IFN-γ secretion, or proliferation as dependent variables and the BCGstrain (+/−zmp1) or immunisation dose (10²-10⁴ CFU) as fixed factors.Prior to the ANOVA, the Levene's test for equality of error varianceswas calculated and if necessary, a power transformation was applied tothe data to meet the equal error variances assumption. The significancelevel was set at 5%.

Example 2 zmp Deletion Increases the Immunogenicity of BCG

To analyse the role of Zmp1 in immunogenicity of BCG, mice wereimmunised with titrated doses of wild type and zmp1 deficient strains.Measuring DTH response upon a footpad challenge with PPD, a differentthreshold for induction was observed for the two vaccine strains. Whilean inoculum of 10³ CFU of the wild type strain was required to producesignificant footpad swelling, one tenth of this dose was sufficient toinduce an equivalent response with the zmp1 mutant, maximal footpadswelling was observed between 10⁴ and 10⁵ CFU (data not shown). Wefurther analysed the degree of footpad swelling at inoculums of 10²-10⁴.As shown in FIG. 1, the zmp1 mutant induced a significantly stronger DTHresponse than did the BCG wild type at 10³ (P=0.0006; Mann Whitney) and10⁴ (P=0.0262) CFU. The experiment was repeated, and a univariateanalysis of variance of the combined data showed that independent of thesize of the inoculum, BCG zmp1 caused a stronger DTH response than thewild type BCG (P<0.001).

The increased immunogenicity of the zmp1 deficient BCG strain wasassociated with increased proliferative and cytokine secreting immuneresponses in vitro of splenocytes from mice immunised with either BCGwild type or BCG zmp1. FIG. 2 illustrates this at an inoculum of 10³CFU, splenocytes from BCG zmp1 immunised mice showed significantly moreIFN-γ production than did the wild type after re-stimulation in vitrowith PPD (P=0.0147 by Mann Whitney) or the M. tuberculosis antigensAg85A (P=0.0133) or TB10.3 (P=0.0005). Similarly, cell proliferation wassignificantly increased in cells from mice immunised with BCG zmp1 (FIG.2). A two-way ANOVA applied on all data, independent of the in vivo BCGdose or the type of antigen used for re-stimulation in vitro, revealedthat the zmp1 mutant caused more efficient IFN-γ secretion (P=0.023) andproliferation (P<0.001) than did wild type BCG.

To analyse whether the IFN-γ secretion derived from CD8 or CD4 positiveT cells, splenocytes from immunised mice were stained and analysed byflow cytometry. Both T-cell subsets were able to produce IFN-γ withfrequencies as high as 7-9% (FIG. 3).

However, when comparing the frequencies of CD8 IFN-γ producing cells inBCG-vaccinated mice to those of untreated mice, the wild type did notsignificantly differ from background levels as analysed by theKruskal-Wallis test with Dunn's multiple comparison test (P>0.1). Incontrast, the BCG zmp1 produced significant levels of IFN-γ for both CD4and CD8 T cells when re-stimulated with either PdBu or PPD (P<0.05). Adirect comparison of the two BCG strains showed that independent of theT-cell subset (CD4 or CD8) or of the re-stimulation antigen (PdBu orPPD), BCG zmp1 caused a strongly increased IFN-γ production.

Example 3 zmp1 Deletion Increases Protective Efficacy of BCG

The protective efficacy of M. bovis BCG zmp1 mutant was tested in amouse model of tuberculosis. Groups of mice (C57BU6) were immunised bysubcutaneous injection of BCG, BCG zmp1 mutant or left untreated. Aftersix weeks mice were challenged with a low dose (10² CFU) of M.tuberculosis H37Rv by aerosol infection and CFU, lung pathology andmortality were monitored. Three weeks after aerosol challenge, lungs ofcontrol mice contained high numbers of M. tuberculosis. Vaccinationreduced the bacterial burden in lungs and in the spleen by approximately1 to 2 logs in the first three weeks of infection. Between three andtwenty-two weeks after challenge mice were able to control theinfection, and CFU counts stabilized.

Twenty-two weeks after infection, lung sections from all three groups ofanimals (unvaccinated control, BCG vaccinated, BCG zmp1 vaccinated)revealed areas of the lung parenchyma with reduced open alveolar space,variable widening of alveolar walls and macrophages filling theairspaces, with levels of non-specific diffuse inflammation affectingthe lung parenchyma being lowest in BCG zmp1 vaccinated mice.Lymphocytic infiltrates, present around blood vessels and injuxtaposition of bronchiolar epithelia, were increased in the vaccinatedgroups compared to the unvaccinated control, and most pronounced in theBCG zmp1 vaccinated animals. On scanning magnification, the lymphocyticinfiltrates in BCG zmp1 mice were the most dense with a tendency toconfluence, while unvaccinated controls and BCG vaccinated animals hadless dense lymphoid infiltrates with only small aggregates. To furtherquantify the extent of the lymphocytic infiltration in the differentgroups of animals, a morphometric approach was chosen, usingAnalySIS-system (Olympus, Switzerland). In each animal lung section, 20lymphocytic aggregates were identified. The mean diameter of lymphoidinfiltrates was measured and the portion of lymphoid infiltrates/lungsection was calculated. The mean diameter in the BCG zmp1 vaccinatedgroup was 0.31 mm compared to 0.13 mm in BCG wt and 0.18 in the controlgroup. Also, the percentage of lymphoid aggregates per lung section wasincreased in BCG zmp1 (7.2%), compared to BCG wt (1.85%) and controlanimals (2.2%).

The biological effect of zmp1 disruption on protective efficacy of BCGwas further investigated in mice by analysis of mean survival times(MST) after infection. The results in FIG. 4 show that unvaccinated micebegan to die 106 days post challenge and all of the unvaccinatedcontrols were dead after 270 days, BCG vaccinated mice began to die byday 189 and the last mouse deceased at day 305, BCG zmp1 immunised micestarted to die at day 244 and the last mouse died at day 364. MST was203.8+/−13.9 days for control mice, 254.8+/−15.7 days for BCG vaccinatedand 297.1+/−15.0 days for BCG zmp1 immunised mice (log rank BCG vs.unimmunised p=0.02; BCG zmp1 vs. BCG p=0.082; BCG zmp1 vs. unimmunisedp=0.00009). Compared to BCG the BCG zmp1 mutant was nearly twice aseffective in prolonging survival (AMST to unvaccinated controls 94 daysfor BCG zmp1 vs. 51 days for BCG); hazard ratios (HR) BCG vs.unimmunised=33% and p=0.032; BCG zmp1 vs. BCG HR=26% and p=0.01; BCGzmp1 vs. unimmunised HR=11% and p=0.000001.

1. Use of at least one live mycobacterium of the M. tuberculosis complexfor preparing a medicament, wherein the function of the zmp1-gene is atleast partially inactivated, preferably inactivated.
 2. Use according toclaim 1, wherein the mycobacterium is M. bovis, preferably M. bovis BCG,or M. tuberculosis.
 3. Use according to claim 1, wherein themycobacterium is non-virulent.
 4. Use according to claim 1, wherein themycobacterium comprises genetic material encoding an antigen and/orimmunogen exogenous or foreign to the mycobacterium.
 5. Use according toclaim 4, wherein the antigen and/or immunogen is selected form the groupconsisting of viral, protozoal, tumor cell-derived, bacterial and fungalantigens and immunogens, preferably selected from the group consistingof antigens and/or immunogens derived from H. pylori, measles virus,mumps virus, rubeola virus, B. burgdorferi, herpes virus, papillomavirus, Pneumococcus spp, tumour cells, leishmania, HIV and SIV.
 6. Useaccording to claim 1 for the prophylaxis and/or treatment of diseases ormedical conditions affected by antigen and/or immunogen expression ofthe mycobacterium.
 7. Use according to claim 1 for the prophylaxisand/or treatment of mycobacterial infections, preferably an infection ofM. tuberculosis.
 8. Use according to claim 1 for the prophylaxis and/ortreatment of a disease or medical condition selected from the groupconsisting of ulcers, measles, mumps, rubeola, lyme disease, herpes,cancer, tetanus, diphtheria, leishmaniasis and AIDS.
 9. A pharmaceuticalcomposition comprising as active substance at least one livemycobacterium of the M. tuberculosis complex, wherein the function ofthe zmp1 -gene is at least partially inactivated, preferablyinactivated, optionally combined with pharmaceutically acceptableexcipients and/or carriers.
 10. Pharmaceutical composition according toclaim 9, wherein the mycobacterium is M. bovis, preferably M. bovis BCG,or M. tuberculosis.
 11. Pharmaceutical composition according to claim 9,wherein the mycobacterium is non-virulent.
 12. Pharmaceuticalcomposition according to claim 9, wherein the mycobacterium comprisesgenetic material encoding an antigen and/or immunogen exogenous orforeign to the mycobacterium.
 13. Pharmaceutical composition accordingto claim 9, wherein the antigen and/or immunogen is selected form thegroup consisting of viral, protozoal, tumor cell-derived, bacterial andfungal antigens and immunogens, preferably selected from the groupconsisting of antigens and/or immunogens derived from H. pylon, measlesvirus, mumps virus, rubeola virus, B. burgdorferi, herpes virus,papilloma virus, tetanustoxin, diphtheriatoxin, Pneumococcus spp, tumourcells, leishmania, HIV and SIV.
 14. Method for the treatment and/orprevention of a disease or medical condition comprising the step ofadministering a pharmaceutical composition according to claim 9 to amammalian, preferably a human patient in need thereof.
 15. Methodaccording to claim 14, wherein the disease and/or medical condition isselected from the group consisting of mycobacterial infections,preferably an infection of M. tuberculosis, ulcers, measles, mumps,rubeola, lyme disease, herpes, tetanus, diphtheria, cancer,leishmaniasis and AIDS.